1. Field of the Invention
The present invention relates to a process for the fermentative production of heterologous proteins using an Escherichia coli strain having an attenuated (p)ppGpp-synthetase II activity.
2. Background Art
The market for recombinant protein pharmaceuticals (pharmaceutical proteins/biologics) has shown strong growth in recent years. Particularly important protein pharmaceuticals are eukaryotic proteins, especially mammalian proteins and human proteins. Examples of important pharmaceutical proteins (pharmaceutically active proteins) are cytokines, growth factors, protein kinase, protein hormones and peptide hormones, and antibodies and antibody fragments. Owing to the costs of producing pharmaceutical proteins, which are still very high, there is a continuous search for more efficient and therefore more cost-effective processes and systems for producing the proteins.
Recombinant proteins are usually produced either in mammalian cell cultures or in microbial systems. Microbial systems have the advantage over mammalian cell cultures in that recombinant proteins can be produced in this way more rapidly and with lower costs. Consequently, bacteria are especially suitable for producing recombinant proteins. The gram-negative enterobacterium Escherichia coli (E. coli) is currently the most frequently used organism for producing recombinant proteins, owing to its very well studied genetics and physiology, short generation time and easy manipulation. Production processes for recombinant proteins in E. coli, which involve the correctly folded target protein being secreted with high yield directly into the fermentation medium are particularly useful.
The literature has disclosed a number of E. coli strains and processes using E. coli strains, by which recombinant proteins are secreted into the fermentation medium. Thus, for example, US2008254511 describes various E. coli strains that have a mutation in the Braun lipoprotein (Lpp) gene and export the overproduced heterologous proteins into the medium.
In order to regulate and coordinate the metabolism under conditions, in which the cell is deficient of amino acids or the energy source, E. coli has a mechanism which is referred to as “stringent control”. In this, the alarmones guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), usually combined as (p)ppGpp hereinbelow, play an important part as regulatory signal molecules, inter alia by slowing down the synthesis of stable RNAs (rRNA, tRNA) and enhancing expression of some amino acid biosynthesis genes, due to an increase in the (p)ppGpp level in the cell (Cashel et al., 1996, Escherichia coli and Salmonella Cellular and Molecular Biology, ed. Neidhardt, F. C. ASM Press, Washington, D.C. pp. 1458-1496). E. coli has two enzymes for synthesizing (p)ppGpp from ATP and GDP (ppGpp) and, respectively, ATP and GTP (pppGpp): (p)ppGpp synthetase I (PSI), encoded by the relA gene, is responsible for (p)ppGpp synthesis under stringent control, when the cells are subject to an amino acid deficiency. However, if cell growth is slowed down due to the primary carbon source being exhausted, (p)ppGPP is synthesized predominantly by (p)ppGpp synthetase II (PSII) which is encoded by the spoT gene. In contrast to PSI, the spoT gene product has also still a (p)ppGpp-3′-pyrophosphohydrolase activity in addition to the (p)ppGpp-synthetase activity, and accordingly can also actively control the cellular (p)ppGpp level by breaking down this compound (Gentry and Cashel, 1996, Mol. Microbiol. 19, 1373-84). The active sites of the two different catalytic activities of the PSII enzyme were shown to be non-identical but nevertheless located in close spatial proximity in the N-terminal region of the protein.
Both E. coli relA mutants and E. coli spoT mutants have been disclosed (Cashel et al., 1996, Escherichia coli and Salmonella Cellular and Molecular Biology, ed. Neidhardt, F. C. ASM Press, Washington, D.C. pp. 1458-1496). Strains having a relA1 mutation, with the IS2 insertion element being integrated in the relA gene sequence (relA::IS2), still possess a small residual PSI activity, whereas relA deletion mutants (ΔrelA) no longer have any PSI activity (Metzger et al., 1989, J. Biol. Chem. 264, 21146-52). In contrast to relA wild-type strains which have a functioning stringent control mechanism (“stringent” phenotype), relA mutants with a reduced or missing PSI activity exhibit a “relaxed” phenotype when encountering an amino acid deficiency situation. The latter phenotype manifests itself inter alia in that the cell is unable to accumulate (p)ppGpp above the basal level, and that the synthesis of stable RNA molecules is not stopped but continues undiminished. The basal level means the (p)ppGpp concentration range (10-30 pmol/A450) for exponentially growing cells under non-limiting growth conditions (i.e. before encountering an amino acid deficiency situation), as disclosed in the literature (Cashel et al., 1996, Escherichia coli and Salmonella Cellular and Molecular Biology, ed. Neidhardt, F. C. ASM Press, Washington, D.C. pp. 1458-1496). Gitter et al. (1995, Appl. Microbiol. Biotechnol. 43, 89-92) have disclosed that the E. coli relA mutant CP79, in comparison with the relA wild-type strain CP78, increasingly releases into the culture medium proteins such as β-lactamase or interferonα1, which have been secreted into the periplasm under artificially induced amino acid deficiency conditions.
Depending on the extent of PSII activity impairment, strains with a mutation in the spoT gene have characteristic (p)ppGpp metabolic defects, inter alia: i) an increased basal ppGpp level during normal (balanced) growth, combined with a lower growth rate, ii) a higher induced ppGpp level during the stringent response, and iii) a lower ppGpp turnover rate after the stringent response has ended.
The DNA sequence of the E. coli spoT gene (SEQ ID NO: 1) codes for the spoT protein with the sequence SEQ ID NO: 2. The spoT gene is expressed as part of an operon which comprises the five genes gmk-rpoZ-spoT-spoU-recG. Expression of the operon and therefore also spoT expression are controlled firstly by the P1 promoter located upstream of the gmk gene, and secondly by the P2 promoter which is located in the gmk gene region coding for the C-terminal moiety (Cashel et al., 1996, Escherichia coli and Salmonella Cellular and Molecular Biology, ed. Neidhardt, F. C. ASM Press, Washington, D.C. pp. 1458-1496). In strains with a spoT1 mutation (SEQ ID NO: 3), the spoT protein has an insertion of the two amino acids glutamine and aspartic acid downstream of the aspartic acid residue 84, and a histidine/tyrosine substitution in position 255 (Durfee et al., 2008, J. Bacteriol. 190, 2597-606). In addition, a number of spoT insertion or deletion mutants have also been described (Xiao et al., 1991, J. Biol. Chem. 266, 5980-90).